人脑源性神经营养因子和神经营养素3真核双表达载体的构建与鉴定

doi:10.3969/j.issn.2095-4344.2014.15.013

中国组织工程研究 ›› 2014, Vol. 18 ›› Issue (15): 2369-2376.doi: 10.3969/j.issn.2095-4344.2014.15.013

• 组织构建与生物活性因子 tissue construction and bioactive factors • 上一篇下一篇

人脑源性神经营养因子和神经营养素3真核双表达载体的构建与鉴定

栗炳南，李卫东，林俊堂，丰慧根

新乡医学院生命科学技术学院，河南省新乡市 453003

出版日期:2014-04-09 发布日期:2014-04-09
通讯作者: 栗炳南, 新乡医学院生命科学技术学院，河南省新乡市 453003
作者简介:栗炳南，男，1983年生，河南省新乡市人，汉族，2011年中南大学湘雅医学院毕业，博士，讲师，目前主要从事神经系统疾病的发病机制与治疗方面的研究。
基金资助:
新乡医学院重点领域招标课题(ZD2011-16)；河南省教育厅科学技术研究重点项目(13A180850)

Construction and identification of bicistronic eukaryotic expression vector of human brain-derived neurotrophic factor and neurotrophine-3

Li Bing-nan, Li Wei-dong, Lin Jun-tang, Feng Hui-gen

Department of Life Sciences and Technology, Xinxiang Medical University, Xinxiang 453003, Henan Province, China

Online:2014-04-09 Published:2014-04-09
Contact: Li Bing-nan, Department of Life Sciences and Technology, Xinxiang Medical University, Xinxiang 453003, Henan Province, China
About author:Li Bing-nan, Ph.D., Lecturer, Department of Life Sciences and Technology, Xinxiang Medical University, Xinxiang 453003, Henan Province, China
Supported by:
the Tender Subject of Key Research Areas of Xinxiang Medical University in 2011, No. ZD2011-16; Key Projects in Scientific Research of Henan Provincial Education Department, No. 13A180850

摘要/Abstract

摘要：

背景：脑源性神经营养因子(brain-derivedneurotrophic factor，BDNF)和神经营养素3 (Neurotrophines-3，NT-3)在细胞分化过程中有重要作用。病毒载体临床应用存在安全隐患，利用真核表达载体表达蛋白为解决安全性问题提供了一种方法。
目的：构建双基因共表达载体pIRES2-BDNF-NT-3并对其进行鉴定。
方法: BDNF和NT-3基因核心序列是通过直接PCR的方法从外周血单个核细胞的基因组DNA中获取。然后将BDNF的cDNA片段插入到pIRES2-EGFP多克隆位点构建pIRES2-BDNF-EGFP载体，随后将NT-3 cDNA片段以替换EGFP的方式插入pIRES2-BDNF-EGFP中，最后构建成为含有内部核糖体进入位点(IRES)的pIRES2-BDNF-NT-3双基因共表达载体。实验通过双酶切和DNA测序方法对其鉴定，将重组的双基因共表达载体感染HEK293细胞，利用RT-PCR与 Western-blot方法检测双基因的表达。
结果与结论：DNA测序显示，提取的BDNF和NT-3均与基因库报道序列一致。构建的pIRES2-BDNF-NT-3双基因共表达载体经Eco RⅠ/Bam HⅠ切出BDNF条带，经Bam HⅠ/NotⅠ双酶切后切出IRES-NT-3片段，经Eco RⅠ/NotⅠ双酶切后切出BDNF-IRES-NT-3片段。RT-PCR与Western-blot方法检测显示，此载体转染后的HEK293细胞均能表达BDNF和NT-3 mRNA和蛋白。结果证实，实验成功采用IRES序列构建了能分别表达的BDNF和NT-3双基因真核表达载体。

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

全文链接：

关键词: 组织构建, 组织工程, 脑源性神经营养因子, 神经营养素3, 真核双表达载体, 转染, 双PCR

Abstract:

BACKGROUND: Human brain-derived neurotrophic factor (BDNF) and neurotrophine-3 (NT-3) are essential genes for cell differentiation. Viral vector has been used numerously in clinical practice, but the security is the most important problem. Eukaryotic expressing vector is a way to solve this question.
OBJECTIVE: To construct and identify pIRES2-BDNF-NT-3 bicistronic eukaryotic expression vector.
METHODS: BDNF and NT-3 genes were obtained from the genomic DNA of human peripheral blood mononuclear cells by PCR. The BDNF cDNA fragment was inserted into the multiple cloning sites of pIRES2-EGFP, to generate the bicistronic eukaryotic expression plasmid pIRES2-BDNF-EGFP. Then NT-3 cDNA fragment was cloned into the pIRES2-BDNF-EGFP, instead of EGFP, to create plasmid pIRES2-BDNF-NT-3. Subsequently pIRES2-BDNF-NT-3 was used to transfect HEK293 cells, and RT-PCR and western blot analysis were applied to test the co-expression of double genes.
RESULTS AND CONCLUSION: The DNA sequencing analysis demonstrated that the BDNF and NT-3 were exactly consistent with the sequence recorded in the GenBank. Enzyme digestion analysis indicated that, in the constructed bicistronic eukaryotic expression vector pIRES2-BDNF-NT-3, BDNF band was obtained by Eco RI /Bam HI digestion, IRES-NT-3 fragment was obtained by Bam HI /Not I digestion, and BDNF-IRES-NT-3 was obtained by Eco RI /Not I digestion. RT-PCR and western blot analysis showed that, after the HEK293 cells were transfected with pIRES2-BDNF-NT-3, double gene was expressed at the mRNA and protein level. Experimental findings suggest that, bicistronic eukaryotic expression vector of BDNF and NT-3 genes can be successfully constructed using IRES sequence.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

全文链接：

Key words: brain-derived neurotrophic factor, nerve growth factors, carrier proteins, tissue engineering

中图分类号:

R318

栗炳南，李卫东，林俊堂，丰慧根. 人脑源性神经营养因子和神经营养素3真核双表达载体的构建与鉴定[J]. 中国组织工程研究, 2014, 18(15): 2369-2376.

Li Bing-nan, Li Wei-dong, Lin Jun-tang, Feng Hui-gen. Construction and identification of bicistronic eukaryotic expression vector of human brain-derived neurotrophic factor and neurotrophine-3[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(15): 2369-2376.

图/表（结果） 4

Amplification of BDNF and NT-3 genes

BDNF and NT-3 genes were obtained from the genomic DNA of human peripheral blood mononuclear cells by PCR. The size of BDNF gene was 744 bp, the size of NT-3 gene was 774 bp (Figure 1A).

Identification of plasmid pIRES₂-BDNF-EGFP

The plasmid pIRES₂-BDNF-EGFP was cut by EcoRI and Bam HI double enzyme. A gene fragment with 744 bp was obtained, which was in full agreed with BDNF gene (NM-001102654.1) (Figure 1B).

Identification of plasmid pIRES₂-BDNF-NT-3 The plasmid pIRES₂-BDNF-NT-3 was cut by Eco RI and Bam HI double enzyme. And a fragment about 744 bp would be obtained. This indicated BDNF gene was inserted into the plasmid pIRES₂-BDNF-NT-3. The plasmid pIRES₂-BDNF-NT-3 was cut by Bam HI and Not I double enzyme. And a fragment about 1 368 bp would be obtained. This indicated IRES-NT-3 gene was inserted into the plasmid pIRES₂-BDNF-NT-3. The plasmid pIRES₂-BDNF-NT-3 was cut by Eco RI and Not I double enzyme. And a fragment about 2 118 bp would be obtained. This indicated that BDNF-IRES-NT-3 gene was inserted into the plasmid pIRES₂-BDNF-NT-3. The sequence of the plasmid pIRES₂-BDNF-NT-3 was in accordance with gene sequence in Gene Bank (Figure 2).

RT-PCR analysis of BDNF and NT-3 mRNA expression in HEK293 cells

To illustrate mRNA expression by pIRES₂-EGFP, pIRES₂-BDNF/EGFP and pIRES₂-BDNF/NT-3 transduced HEK293 cells, we evaluated the expression of BDNF and NT-3 by RT-PCR analysis. RT-PCR was performed using BDNF-specific primers and the β-actin sequence served as an internal standard. GFP expression was monitored in pIRES₂-EGFP and pIRES₂-BDNF/EGFP transduced HEK293 cells by inverted fluorescence microscopy. Expression of BDNF mRNA was higher in either pIRES₂-BDNF/EGFP or pIRES₂-BDNF/NT-3 transduced HEK293 cells than that pIRES₂-EGFP transduced HEK293 cells or negative control (P < 0.01; Figure 3A, C). As shown above, expression of NT-3 mRNA was higher in pIRES₂-BDNF/NT-3-transduced HEK293 cells than the other three (P < 0.01; Figure 3B, D). These results demonstrated that the BDNF and NT-3 had been introduced successfully into HEK293 cells by pIRES₂-BDNF/EGFP and pIRES₂-BDNF/NT-3.

Western blot analysis of BDNF and NT-3 expression in HEK293 cells

To illustrate protein expression by pIRES₂-EGFP, pIRES₂-BDNF/EGFP, and pIRES₂-BDNF/NT-3 transduced HEK293 cells, we evaluated the expression of BDNF and NT-3 by western blot assay. After 72 hours of transduction, we collected the culture supernatants of infected HEK293 cells without serums which were processed for western blot analysis using an anti-BDNF and anti-NT-3 antibody.

The β-actin served as an internal standard. The results suggested that exogenous BDNF protein was strongly expressed in pIRES₂-BDNF/EGFP and pIRES₂-BDNF/NT-3 transduced HEK293 cells, but the pIRES₂-EGFP transduced HEK293 cells and no transduced expression of endogenous BDNF was very low (P < 0.01; Figure 4A, C). As shown above, western blot analysis also revealed that exogenous NT-3 protein was strongly expressed in pIRES₂-BDNF/NT-3 transduced HEK293 cells, and the pIRES₂-EGFP, pIRES₂-BDNF/EGFP transduced HEK293 cells expression of endogenous NT-3 was very low (P < 0.01; Figure 4B, D). These results also demonstrated that BDNF and NT-3 had been introduced successfully into HEK293 cells by pIRES₂-BDNF/NT-3.

参考文献

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引言

Spinal cord injury is now a major problem plaguing the medical profession^[1] . In recent years, the discovery of neurotrophic factors brings new hope in the treatment of spinal cord injury^[2] . Neurotrophic factors, which can effectively promote and maintain the nerve cell growth, survival, differentiation and the execution of a function^[3-5] , is originally extracted from the snake venom. In 1980s, Barde et al found neurotrophic growth factors, with the structure similar to the nerve growth factor, in the brain extraction liquid, and named brain-derived neurotrophic factor (BDNF)^[6-8] . BDNF is widely distributed in the brain, and the action scope is very wide. In the development process, BDNF is a neurotrophic promoting factor that plays an important role in maintaining neuron survival, improving neuron differentiation and being an indispensable factor in myelination^[9-12] . Neurotrophines-3 (NT-3) is a new neurotrophic factor found through studying the homology of sequence between nerve growth factor and BDNF by Ernfors et al in 1990, which is one member of neurotrophin family or nerve growth factor family^{[13-15] .} The distribution of NT-3 and its biological activity in a variety of distinct groups of neurons is significantly different from nerve growth factor and BDNF. NT-3 has its characteristic function such as maintaining the survival of neurons in vivo, promoting nerve cell differentiation and inducing neurite growth^[16-18] .

In this study, we attempts to construct a co-expression plasmid on a double gene consisting of BDNF and NT-3, and then transfected HEK293 cells to detect the expression. This study will provide experimental basis for the next experiment addressing co-transfected mesenchymal stem cells, improving the internal environment of spinal cord injury, regulating differentiation direction about mesenchymal stem cells, increasing the number of newborn neurons, and promoting functional recovery after spinal cord injury.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

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材料方法

Design

A genetic study.

Time and setting

The experiment was completed in the Life Science College of Xinxiang Medical University, China from November 2012 to August 2013.

Materials

Fresh anticoagulant blood are donated from a volunteer in this project group who is a male, 30 years old, with no disease previously, in good general situation, and there is no diseases after physical examination. The donor has been informed of the objective, significance and possible discomfort of this experiment, and he signed the informed consent, we have got the permission of this volunteer. Escherichia coli DH5α are kept in Stem Cells and Biological Treatment Center at Xinxiang Medical University, China.

Vector

pIRES₂-EGFP plasmid was purchased from Beijing TianDz Inc., Beijing, China.

Cells

HEK293 cells are kept in Stem Cells and Biological Treatment Center at Xinxiang Medical University, China.

Methods

Design of the primers

BDNF (NM-001102654.1) and NT-3 (NM-001102654.1) gene sequence in Gene Bank was used as the template to design the primers. The coding sequence of human BDNF gene fragment was totally on the exon2 of the genomic DNA. Two primers were designed to amplify the human BDNF. Eco RI restriction enzyme cut site and protective base were added in the upstream prime, and Bam HI restriction enzyme cut site and protective base were added in the downstream primer, the length of amplified fragment was 744 bp.

Two primer pairs were designed to amplify human NT-3 from the genomic DNA. Forward-long primer was 4 bases longer than forward-short at the 5' end. So did primer reverse-long than reverse-short. The underlined bases in forward primers were introduced parts of sequence of Bst XI site, and in reverse primers of Not I site, which will be used to assemble the Bst XI and Not I sticky ends. The length of amplified fragment was 774 bp.

Construction of pIRES₂-BDNF-EGFP

The genomic DNA of human peripheral blood mononuclear cells severed as the template and the primer of BDNF was used to amplify BDNF gene. The coding sequence of human BDNF gene fragment was totally on the exon2 of the genomic DNA. A total of 20 μL PCR reaction system was added, including the genomic DNA

0.5 μL (10 ng), 2 × Prime STAR Max DNA Polymerase 10 μL, RNase-Free Water 7.5 μL, upstream primer and downstream primer 2 μL. The reaction conditions were: 95 ℃ for 5 minutes, 98 ℃ for 10 seconds, 55 ℃ for

5 seconds, and 72 ℃ for 5 seconds, 30 cycles, finally, maintained at 72 ℃ for 5 minutes. PCR product was purified according to the PCR Purification Kit, then the PCR product and plasmid pIRES₂-EGFP was cut by Eco RI and Bam HI. After digested, PCR product was purified by the PCR Purification Kit. The DNA fragment was inserted into the plasmid by T4 DNA ligase, 20 μL system was added, including pIRES₂-EGFP 2 μL, cDNA (BDNF)

8 μL, T4 DNA ligase 0.2 μL was added at 22 ℃ for 30 minutes. The DNA fragment was translated into Escherichia coli DH5α and placed on LB plate (kan^r) at

37 ℃ in the incubator for 16 hours. Monoclonal colony was picked up and shaken for 12-16 hours at 37 ℃ with a speed of 225 r/min. The plasmid was extracted and identified by using EcoRI and Bam HI double enzyme digestion. The recombinant plasmid pIRES₂-BDNF-EGFP was obtained.

PCR amplification of NT-3

Two parallel PCRs were set up using either forward-long/ reverse-long or forward-short/reverse-short primer pairs, with the genomic DNA of human peripheral blood mononuclear cells as templates. Both amplifications were subjected to 30 cycles at 94 ℃ for 30 seconds, 56 ℃ for 30 seconds and 72 ℃ for 1 minute, followed by a 5 minute extension at 72 ℃ using Prime STAR Max DNA Polymerase. The expected DNA fragments in the four PCR products were puri?ed separately using DNA gel extraction kit and quanti?ed by Nano Drop 2000 UV-Vis Spectrophotometers.

Construction of pIRES₂-BDNF/NT-3 vector

pIRES₂-BDNF-EGFP was digested with Bst XI and Not I, followed by phenol: chloroform extraction and ethanol precipitation. Meanwhile, each pooled NT-3 PCR products ampli?ed with Prime STAR Max DNA Polymerase were mixed, denatured at 94 ℃ for 4 minutes and re-annealed at 65 ℃ for 2 minutes. For a ligation reaction, the annealed NT-3 and linearized pIRES2-BDNF-EGFP (molar ratio equal to 4:1) were incubated with T4 DNA ligase at 16 ℃ for 3 hours. The two ligation products were used to transform Escherichia coli DH5a CaCl₂ competent cells following standard method. Screening of the transformants was performed by Bst XI plus Not I digestion following alkalinelysis plasmid preparation, and the recombinants were con?rmed by DNA sequencing.

Two parallel PCRs were set up using either forward-long/reverse-long (PCR products A) or forward-short/reverse-short primer pairs (PCR products B), with the genomic DNA of human peripheral blood mononuclear cells as templates. Equimolar of the two PCR products A and B were mixed, heat denatured and re-annealed to generate hybridized DNA fragments. Four types of double-stranded DNA molecules with equal proportions are generated via random complementary pairing. They are molecule I, II, III and IV. Two of them, Ⅰ and II, are blunt-ended and another two are sticky-ended. The molecule III possess a 5' Bst XI sticky end and a 3' Not I sticky end, which allow the fragment to be cloned directionally into pIRES₂-BDNF-EGFP digested with Bst XI and Not I.

In vitro transduction of HEK293 cells

HEK293 cells were maintained in Dulbecco’s modified Essential Medium (DMEM) containing 10% newborn bovine serum and 100 mg/L penicillin/streptomycin. Cells were maintained in a humidified environment at 37 ℃ and 5% CO₂. Cell viability was monitored with trypan blue exclusion method. The viability was over 95% in all experiments. Cells were seeded at a density of 5 × 10⁵ cells/well in a six-well tissue culture plate and cultured for 24 hours to reach 60%-80% confluence. HEK293 cells were either mock infected or infected with the pIRES₂-EGFP, pIRES₂-BDNF/EGFP and pIRES₂-BDNF/NT-3 vectors using Lipofectamine™ 2000 respectively for 2 hours at 37 ℃ at 5 μg per well. Two hours later, the transfection medium was removed and fresh complete growth medium was added. Twenty-four hours post-transfection, they were observed under the inverted fluorescent microscope. Analysis of the BDNF and NT-3 gene expression by RT-PCR and western blot analysis was performed 3 days later.

RT-PCR analysis of BDNF and NT-3 expression in HEK293 cells

To detect the BDNF and NT-3 mRNA expression levels in HEK293 cells which were either mock infected or infected with the pIRES₂-EGFP, pIRES₂-BDNF/EGFP, and pIRES₂-BDNF/NT-3 vectors respectively, the BDNF and NT-3 expression was primarily assessed by means of RT-PCR. The collected cells were added per 1 mL TRIZOL reagent and pipetting repeatedly, then total cellular RNA was extracted in accordance with the instructions, the extracted total RNA was transcribed reversely into cDNA. Then reverse transcription of the cDNA was amplified with the PCR, and the reaction conditions were pre-denaturation under 94 ℃ for 5 minutes; 94 ℃ for 30 seconds, 55 ℃ for 30 seconds, 72 ℃ for

30 seconds, total 35 cycles, followed by extension at 72 ℃ for 10 minutes. 5 μL PCR amplification products were then added into 1 μL loading buffer, then subject to 20 g/L agarose gel for electrophoresis under 100 V voltage. The image was analyzed using gel imaging system, and the gray value of each band was calculated.

Western blot analysis of BDNF and NT-3 expression in HEK293 cells

A standard western blot protocol was used to detect BDNF and NT-3 protein expression in HEK293 cells which were either mock infected or infected with the pIRES₂-EGFP, pIRES₂-BDNF/EGFP and pIRES₂-BDNF/NT-3 vectors respectively 3 days later. We collected the culture supernatants of infected HEK293 cells without serums which were processed for western blot analysis using an anti-BDNF and anti-NT-3 antibody. The 20 μg protein extracted from culture supernatants of transduced HEK293 cells was separated on SDS-PAGE under reducing and denaturing conditions and transferred to polyvinylidene difluoride membrane. The membranes were incubated in polyclonal rabbit anti-BDNF antibody (1∶500, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or polyclonal rabbit anti-NT-3 antibody (1∶250, Santa Cruz Biotechnology), polyclonal rabbit anti-β-actin

(1∶500, Santa Cruz Biotechnology) was used as the loading control. After washing with TBS for three times

(10 minutes per time), they were incubated with horseradish peroxidase conjugated goat anti-rabbit lgG (1:5 000) at room temperature for 2 hours. After three TBS washing (with 15 minutes per time), the cells were observed with X-ray films. The amount of signal was quantified with Gel-Pro analyzer software.

Main outcome measures

The mRNA and protein expressions of NT-3 and BDNF were detected by RT-PCR and western blot assay, respectively.

Statistical analysis

Experimental data were analyzed using SPSS 13.0 statistical software (SPSS, Chicago, IL, USA) and the results are expressed as mean±SD. Data comparisons between groups were tested by one-way analysis of variance, P < 0.05 was considered statistically significant

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

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讨论

To improve the cloning efficiency and simplify the cloning procedure, we developed twin PCR to create insert DNA with single-stranded overhangs at the ends which were complimentary to restriction enzyme digested vector^[15]. As shown in Figure 1, PCR products reassembled via complimentary pairing after heat-denaturing and re-annealing. Four types of molecules with equal proportions were created, two of which were blunt-ended and another two were sticky-ended. Theoretically, half of the sticky-ended products are suitable for a certain ligation. Using twin PCR strategy, hybridized PCR products flanked with all kinds of sticky ends that are compatible with almost all restriction enzymes can be generated based solely on primer designing and PCR amplification. Since no restriction enzyme digestion is required for the PCR products, twin PCR overcomes the problems caused by end sensitivity of restriction enzymes, and the internal restriction sites within the amplified sequences will not limit the selection of cloning sites. This is particularly useful for cloning long PCR products, which have more opportunities to contain kinds of restriction enzymes sites.

In this article, NT-3 was directly inserted into pIRES2-BDNF-EGFP at the Bst XI and Not I sites. Those more efficient and economical enzymes, such as Bst XI and Not I, could always be chosen for vector digestion. It is not any longer necessary to buy so many kinds of restriction enzymes for cloning. Meanwhile, our cloning method has other advantages. It is so simple that the whole procedure, including PCR, enzyme digestion, ligation and transformation could be completed in 8 hours, leading to the substantial saving in time, cost and labor. In addition, interested inserts are amplified using proof-reading DNA polymerase, which provides a faithful guarantee for the PCR products. These advantages make it a universal technique for the directional cloning of PCR products.

Neurotrophic factors work through its receptors, the different receptors mediated different biological effects. Due to different distribution of receptors, different neurotrophic factors have different functions which functioning on different neurons. A neuron can contain different receptors of neurotrophic factors at the same time. When different nutritional factors act on the same neuron, they can make up for each other in functions.

BDNF can promote the development and differentiation of embryonic neurons, maintain the survival of a variety of neurons, and improve the biological activity of neurons, it also can reduce the natural death of neurons after spinal cord injury^[16-20] . Study found that BDNF can prevent neuronal apoptosis by tyrosine receptor signal pathway^[21] . Meanwhile BDNF also has nutritious effect on peptidergic neurons and GABA neurons[^22] . BDNF can promote neural stem cells differentiated directionally into neurons in vitro. Under the conditions in vivo, BDNF contributes to promote the survival, differentiation, migration of exogenous neural stem cells, and it can replace and repair the nerve conduction bundle of spinal cord, so this is beneficial to the functional recovery of the injured spinal cord^[23] . BDNF can regulate the growth and survival of neurons, react on the rubrospinal tract and red nucleus neurons, also promote the regeneration and functional recovery of rubrospinal tract after spinal cord injury^[24] . Experimental studies using BDNF gene modified neural stem cell transplantation for treatment of spinal cord injury in animal models found that BDNF has the protective effect on red nucleus neurons^[25] . In addition, BDNF promotes the growth of corticospinal axons and it is benefit for a partial nerve recovering its function after spinal cord injury, the same time it will slow down the death rate of injured neurons obviously^[26] .

NT-3 may play an important role in promoting the growth of neurons^[27] . NT-3 is the strongest neurotrophic factor in the spinal cord^[28] . After oligodendrocyte precursor cells are transfected by NT-3, the NT-3 can promote precursor cells differentiation into oligodendrocytes, and increase the number of myelin basic protein secretion, as well as help the co-cultured neurons form myelin sheath. After spinal cord injury occurs, the expression of NT-3 is increased sharply, and has a protective effect on neurons, at the same time it reduces spinal cord injury^[29-30] . NT-3 can protect the neurons by influencing superoxide dismutase and malondialdehyde expression after spinal cord injury[^31] . After spinal cord injury, exogenous NT-3 can promote the regeneration of axons^[32] .

By gene recombination we successfully constructed eukaryotic expression vector of BDNF and NT-3, and these two genes can be co-expressed in HEK293 cells. Checking and analyzing the national and international relative literatures, there is no study about gene therapy by using BDNF and NT-3 to promote the differentiation of mesenchymal stem cells following spinal cord injury, so this experiment will provide new methods for animal internal experiments in the next step.

In this study, BDNF and NT-3 were successfully inserted into a bicistronic eukaryotic expression vector and a plasmid pIRES2-BDNF-NT-3 was constructed successfully. We plan to transfect this plasmid expressed by double genes into mesenchymal stem cells in the next experiment, and to achieve the stable transfected cell lines. We surmised that in promoting neuronal regeneration, mesenchymal stem cells modified by the double genes will be more significant compared with the single gene modified cells.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

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人脑源性神经营养因子和神经营养素3真核双表达载体的构建与鉴定

Construction and identification of bicistronic eukaryotic expression vector of human brain-derived neurotrophic factor and neurotrophine-3

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